Size matters: Large copy number losses in Hirschsprung disease patients reveal genes involved in enteric nervous system development

Hirschsprung disease (HSCR) is a complex genetic disease characterized by absence of ganglia in the intestine. HSCR etiology can be explained by a unique combination of genetic alterations: rare coding variants, predisposing haplotypes and Copy Number Variation (CNV). Approximately 18% of patients have additional anatomical malformations or neurological symptoms (HSCR-AAM). Pinpointing the responsible culprits within a CNV is challenging as often many genes are affected. Therefore, we selected candidate genes based on gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics. Next, we used a zebrafish model to investigate whether loss of these genes affects enteric neuron development in vivo. This study included three groups of patients, two groups without coding variants in disease associated genes: HSCR-AAM and HSCR patients without associated anomalies (HSCR-isolated). The third group consisted of all HSCR patients in which a confirmed pathogenic rare coding variant was identified. We compared these patient groups to unaffected controls. Predisposing haplotypes were determined, confirming that every HSCR subgroup had increased contributions of predisposing haplotypes, but their contribution was highest in isolated HSCR patients without RET coding variants. CNV profiling proved that specifically HSCR-AAM patients had larger Copy Number (CN) losses. Gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics were used to determine plausible candidate genes located within CN losses. Validation in zebrafish using CRISPR/Cas9 targeting confirmed the contribution of UFD1L, TBX2, SLC8A1, and MAPK8 to ENS development. In addition, we revealed epistasis between reduced Ret and Gnl1 expression and between reduced Ret and Tubb5 expression in vivo. Rare large CN losses—often de novo—contribute to HSCR in HSCR-AAM patients. We proved the involvement of six genes in enteric nervous system development and Hirschsprung disease.

Gut innervation and enteric nervous system development: a spatial, temporal and molecular tour de force

ABSTRACTDuring embryonic development, the gut is innervated by intrinsic (enteric) and extrinsic nerves. Focusing on mammalian ENS development, in this Review we highlight how important the different compartments of this innervation are to assure proper gut function. We specifically address the three-dimensional architecture of the innervation, paying special attention to the differences in development along the longitudinal and circumferential axes of the gut. We review recent information about the formation of both intrinsic innervation, which is fairly well-known, as well as the establishment of the extrinsic innervation, which, despite its importance in gut-brain signaling, has received much less attention. We further discuss how external microbial and nutritional cues or neuroimmune interactions may influence development of gut innervation. Finally, we provide summary tables, describing the location and function of several well-known molecules, along with some newer factors that have more recently been implicated in the development of gut innervation.